It is often desirable to administer drugs using controlled- or sustained-release formulations that can maintain therapeutic blood levels of the drug over extended periods of time. These controlled release formulations reduce the frequency of dosing, for enhanced convenience and compliance, and also reduce the severity and frequency of side effects. By maintaining substantially constant blood levels and avoiding blood level fluctuations of the drug, such as are associated with conventional immediate release formulations that are administered several times a day, controlled- or sustained-release formulations can provide a better therapeutic profile than is obtainable with conventional immediate release formulations.
Known methods for controlled- or sustained-drug release include implanted devices, such as osmotic pumps, and drug dispersed in a biocompatible polymer matrix, which can be implanted, administered orally, or injected. Examples of biocompatible polymers used in such applications include poly(lactic acid) and poly(lactic acid-co-glycolic acid). The polymer typically undergoes slow hydrolysis in vivo to continually release the entrapped drug over time. The polymer degradation products are non-toxic and absorbed or metabolized by the body. For example, when the biocompatible polymer is poly(lactic acid) or poly(lactic acid-co-glycolic acid), the degradation products are the parent acids, lactic acid and glycolic acid, which are absorbed by the body.
International published application WO 03/034988 discloses compositions of a salt of a pharmacologically active compound and a lipophilic counterion and a pharmaceutically acceptable water soluble solvent that are combined together to provide an injectable composition. When injected into an animal at least a part of the composition precipitates to form a depot that slowly releases the pharmacologically active compound over time.
U.S. patent application no. US 2004/0220264 discloses compositions, methods of making the compositions, and uses of compositions that include a molecular complex between an acidic pharmaceutical drug and a functional substance. The functional substance can be an alkaline amino acid, an amino acid amide, an amino acid ester, or a related amino acid. The compositions are allegedly useful for delivering the drug into cutaneous tissue.
U.S. patent application no. US 2004/0197408 discloses formulations of a diblock copolymer having a hydrophobic block and hydrophilic block, an additive selected from an amino acid, and an oligopeptide. The formulations, when admixed with water, form drug delivery vehicles in micellar form.
Oligonucleotides are small double-stranded or single-stranded segments of DNA or RNA, typically about 20-30 nucleotide bases in length. Oligonucleotides can be synthetic or natural, and bind to a particular target molecule, such as a protein, metabolite, or other nucleic acid sequence. Oligonucleotides are a promising class of therapeutic agents currently in pre-clinical and clinical development for treating a variety of diseases and disorders. Like biologics, e.g., peptides or monoclonal antibodies, oligonucleotides are capable of binding specifically to molecular targets and, through binding, inhibiting target function. Oligonucleotides include for example, siRNA and aptamers.
SiRNA are small strands of RNA that interfere with the translation of messenger RNA. SiRNA can be double stranded or single stranded. Generally, double stranded siRNA works better than single stranded siRNA. Typically, siRNA are about 20 to 25 nucleotides long. SiRNA can be used to interfere with the expression of genes. They bind to the complementary portion of the target messenger RNA and tag it for degradation. SiRNA's effect of inhibiting gene expression is commonly known as gene “silencing.” The siRNA causes the destruction of messenger RNA that shares sequence homology with the siRNA to within one nucleotide resolution (Elbashir S. M. et al, Genes Dev., 15 (2001) 188-200). It is believed that the siRNA and the targeted mRNA bind to an “RNA-induced silencing complex” or “RISC,” which cleaves the targeted mRNA. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. The siRNA mediated degradation of a mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
The ability to specifically inhibit expression of a target gene by siRNA has obvious benefits. For example, many diseases arise from the abnormal expression of a particular gene or group of genes. SiRNA can be used to inhibit the expression of the deleterious gene and therefore alleviate symptoms of a disease or even provide a cure. For example, genes contributing to a cancerous state or to viral replication could be inhibited. In addition, mutant genes causing dominant genetic diseases such as myotonic dystrophy could be inhibited. Inflammatory diseases such as arthritis could also be treated by inhibiting such genes as cyclooxygenase or cytokines. Examples of targeted organs include, but are not limited to the liver, pancreas, spleen, skin, brain, prostrate, heart. In addition, siRNA could be used to generate animals that mimic true genetic “knockout” animals to study gene function. Useful sequences of siRNA can be identified using known procedures such as described in Pharmacogenomics, 6(8):879-83 (December 2005), Nat. Chem. Biol., 2(12):711-9 (December 2006), Appl Biochem. Biotechnol., 119(1): 1-12 (October 2004), U.S. Pat. No. 7,056,704 and U.S. Pat. No. 7,078,196).
Aptamers, are oligonucleotides that bind to a particular target molecule, such as a protein or metabolite. Typically, the binding is through interactions other than classic Watson-Crick base pairing. A typical aptamer is 10-15 kDa in size (i.e., 30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates among closely related targets (e.g., will typically not bind other proteins from the same gene family) (Griffin, et al. (1993), Gene, 137(1): 25-31; Jenison, et al. (1998), Antisense Nucleic Acid Drug Dev., 8(4): 265-79; Bell, et al. (1999), In Vitro Cell. Dev. Biol. Anim., 35(9): 533-42; Watson, et al. (2000), Antisense Nucleic Acid Drug Dev., 10(2): 63-75; Daniels, et al. (2002), Anal. Biochem., 305(2): 214-26; Chen, et al. (2003), Proc. Natl. Acad. Sci. U.S.A., 100(16): 9226-31; Khati, et al. (2003), J. Virol., 77(23): 12692-8; Vaish, et al. (2003), Biochemistry, 42(29): 8842-51).
Aptamers can be created by an entirely in vitro selection process (Systematic Evaluation of Ligands by Experimental Enrichment, i.e., SELEX™) from libraries of random sequence oligonucleotides as described in U.S. Pat. Nos. 5,475,096 and 5,270,163. Aptamers have been generated against numerous proteins of therapeutic interest, including growth factors, enzymes, immunoglobulins, and receptors (Ellington and Szostak (1990), Nature, 346(6287): 818-22; Tuerk and Gold (1990), Science, 249(4968): 505-510).
Aptamers have a number of attractive characteristics for use as therapeutics. In addition to high target affinity and specificity, aptamers have shown little or no toxicity or immunogenicity in standard assays (Wlotzka, et al. (2002), Proc. Natl. Acad. Sci. U.S.A., 99(13): 8898-902). Indeed, several therapeutic aptamers have been optimized and advanced through varying stages of pre-clinical development, including pharmacokinetic analysis, characterization of biological efficacy in cellular and animal disease models, and preliminary safety pharmacology assessment (Reyderman and Stavchansky (1998), Pharmaceutical Research, 15(6): 904-10; Tucker et al., (1999), J. Chromatography B., 732: 203-212; Watson, et al. (2000), Antisense Nucleic Acid Drug Dev., 10(2): 63-75).
Oligonucleotides, to be effective, must be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen. In addition, siRNA, to be effective, must enter the cell. Aptamers, however, are directed against extracellular targets and, therefore, do not suffer from difficulties associated with intracellular delivery.
It is important, however, that the pharmacokinetic properties for all oligonucleotide-based therapeutics be tailored to match the desired pharmaceutical application. Early work on nucleic acid-based therapeutics has shown that, while unmodified oligonucleotides are degraded rapidly by nuclease digestion, protective modifications at the 2′-position of the sugar, and use of inverted terminal cap structures, e.g., [3′-3′dT], dramatically improve nucleic acid stability in vitro and in vivo (Green, et al. (1995), Chem. Biol., 2(10): 683-95; Jellinek, et al. (1995), Biochemistry, 34(36): 11363-72; Ruckman, et al. (1998), J. Biol. Chem., 273(32): 20556-67; Uhlmann, et al. (2000), Methods Enzymol., 313: 268-84). For example, in some SELEX selections (i.e., SELEX experiments or SELEX ions), the starting pools of nucleic acids from which aptamers are selected are typically pre-stabilized by chemical modification, for example by incorporation of 2′-fluoropyrimidine (2′-F) substituted nucleotides, to enhance resistance of the aptamers against nuclease attack. Aptamers incorporating 2′-O-methylpurine (2′-OMe purine) substituted nucleotides have also been developed through post-SELEX modification steps or, more recently, by enabling synthesis of 2′-OMe-containing random sequence libraries as an integral component of the SELEX process itself.
In addition to clearance by nucleases, oligonucleotide therapeutics are subject to elimination via renal filtration. As such, a nuclease-resistant oligonucleotide administered intravenously exhibits an in vivo half-life of <10 min, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood stream into tissues or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus. Conjugation to a PEG polymer (“PEGylation”) can dramatically lengthen residence times of oligonucleotides in circulation, thereby decreasing dosing frequency and enhancing effectiveness against targets. Previous work in animals has examined the plasma pharmacokinetic properties of PEG-conjugated aptamers (Reyderman and Stavchansky (1998), Pharmaceutical Research, 15(6): 904-10; Watson, et al. (2000), Antisense Nucleic Acid Drug Dev., 10(2): 63-75)). Determining the extravasation of an oligonucleotide therapeutic, including oligonucleotide therapeutics conjugated to a modifying moiety or containing modified nucleotides and, in particular, determining the potential of oligonucleotides or their modified forms to access diseased tissues (for example, sites of inflammation, or the interior of tumors) define the spectrum of therapeutic opportunities for oligonucleotide intervention.
Typically, therapeutic oligonucleotides are administered by injection, for example, by subcutaneous or intravenous injection. Accordingly, the oligonucleotides must be dissolved or dispersed in a liquid vehicle for administration. The relatively high molecular weight of oligonucleotides, and in particular oligonucleotides that have been derivatized, for example by PEGylation, however, often makes it difficult to obtain a pharmaceutical composition wherein the oligonucleotide is dissolved or dispersed in a pharmaceutically acceptable solvent at a sufficient concentration to provide a pharmaceutical composition that is clinically useful for administration to an animal.
U.S. published application no. 2005/0175708 discloses a composition of matter that permits the sustained delivery of aptamers to a mammal. The aptamers are administered as microspheres that permit sustained release of the aptamers to the site of interest so that the aptamers can exert their biological activity over a prolonged period of time. The aptamers, can be anti-VEGF aptamers.
P. Burmeister et al., (2004), Chemistry and Biology: 15, 25-33 disclose a method for generating a 2′-O-methyl aptamer (ARC245) that binds to vascular endothelial growth factor, which exhibits good stability.
There remains a need in the art, however, for therapeutic agent containing pharmaceutical compositions, suitable for injection or implantation, wherein the formulation provides controlled- or sustained-release of the therapeutic agent. There is also a need in the art for improved pharmaceutical compositions, wherein the therapeutic agent is an oligonucleotide. In particular, there is a need for pharmaceutical composition wherein the oligonucleotide can be dissolved or dispersed in a pharmaceutically acceptable solvent at a sufficient concentration to provide a pharmaceutical composition that is clinically useful for administration to an animal, and, in particular, administration by injection. The present invention addresses this as well as other needs.
Citation of any reference in Section 4 of this application is not to be construed that such reference is prior art to the present application.